US8609353B2 - Diagnostics and methods for removal and detection of interferents - Google Patents

Diagnostics and methods for removal and detection of interferents Download PDF

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US8609353B2
US8609353B2 US12/160,610 US16061007A US8609353B2 US 8609353 B2 US8609353 B2 US 8609353B2 US 16061007 A US16061007 A US 16061007A US 8609353 B2 US8609353 B2 US 8609353B2
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sample
peptide
interferent
prior
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US20090304778A1 (en
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Mitchell C. Sanders
Diane L. Ellis-Busby
Jennifer M. Havard
James C. Comolli
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Woundcheck Laboratories (US) Inc
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Systagenix Wound Management US Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • the color substrates such as TMB (3,3′,5,5′-tetramethyl benzidine), ABTS (2,2′azino-bis-(3-ethylbenzthiazoline-6-sulfionic acid), DAB (3,3′-diaminobenzidine tetrahydrochloride), NBT (nitro-blue tetrazolium chloride), and BCIP (5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt) used with these enzymes are commercially available in both soluble and insoluble forms, with various applications in immunochemistry, enzyme assays, and diagnostics. It would be helpful to remove and detect interferents which hinder the efficiency of these applications.
  • This invention encompasses the use of a cation exchange material, such as sulfopropyl (SP) or carboxylmethyl (CM), that can be used at a slightly basic pH (about 7.5-about 8.0) to remove elastase (e.g., human elastase) from wound samples or other specimens (including but not limited to blood, urine, stool or tissue biopsies) that may interfere with any diagnostic assay, including one that is antibody, enzyme, or PCR based.
  • SP sulfopropyl
  • CM carboxylmethyl
  • the cation exchange material can be used in multiple formats (including, but not limited to, beads, membranes, non-woven fibers and/or cotton swabs, sponges, filters, wipes, pads, and dressings) that can remove elastase within seconds prior to the sample being loaded into a lateral flow chamber, dipstick, or any kind of liquid assay including, but not limited to, ELISA, and lateral flow or push through assays in which the colored substrate is soluble or insoluble. In some embodiments, every trace of human elastase is removed.
  • the material can be placed on, for example, a dressing, pad, or wipe to be used to remove elastase in situ, which may improve wound healing by removing deleterious enzymes, including host enzymes, that prevent healing.
  • this invention includes a method of removing at least one interferent from a sample comprising contacting a cation exchange material and/or phosphocellulose to said sample, thereby removing said interferent.
  • this invention includes a method of increasing the specificity of an assay of a sample, said sample comprising at least one interferent, comprising contacting a cation exchange material and phosphocellulose to said sample, thereby removing said interferent.
  • the interferent is selected from the group consisting of peroxidases, elastase, papain, matrix metalloproteases, heme and viruses.
  • the sample is selected from the group consisting of wound, tissue, urine, saliva, blood, and stool samples.
  • the sample is selected from the group consisting of fluid, biopsy and solid samples.
  • said interferent is peroxidase
  • the phosphocellulose is contacted to said sample to remove specifically said peroxidase.
  • said interferent is hemoglobin
  • the phosphocellulose is contacted to said sample to remove specifically the hemoglobin
  • the phosphocellulose is selected from the group consisting of p11 resin and p81 paper.
  • the interferent is elastase
  • the cation exchange material is contacted to said sample to remove specifically the elastase.
  • the cation exchange material is selected from the group consisting of sulfopropyl sepharose and carboxylmethyl sepharose.
  • the elastase is capable of detection by a peptide comprising the amino acid sequence of PFPQANYITY (SEQ ID NO: 1).
  • the papain is capable of detection by a peptide comprising the amino acid sequence of PMPPLCTSM (SEQ ID NO: 2).
  • the assay is selected from the group consisting of an enzyme-based assay, an immunoassay, an immunostaining application, an antibody-based assay, a PCR-based assay, a protease-based assay, a diagnostic test, a push through assay and a lateral flow assay.
  • the interferent is removed prior to an immunostaining or a PCR reaction.
  • the cation exchange material or phosphocellulose is applied to a gauze, cleansing wipe, bead, glass frit, swab, wipe, pad, membrane, a non-woven fiber, a sponge, a filter, a biopsy punch or a dressing prior to contact to said sample.
  • the cation exchange material or phosphocellulose is incorporated into a biopsy punch device prior to contact to the sample.
  • the cation exchange material or phosphocellulose is applied to a lateral flow assay device prior to contact to the sample.
  • the cation exchange material or phosphocellulose is applied to the back of a swab (e.g., a cotton, polyurethane, or polyester swab), a medical device (e.g., a speculum) or sampling device,
  • a sampling device is any device used to acquire a sample from a person (e.g., a patient), such as a fluid or tissue sample.
  • a fluid or tissue sample e.g., a patient
  • a wound biopsy device e.g., a wound biopsy device.
  • said cation exchange membrane or phosphocellulose is incorporated into a collection container, a collection device, a tube, a vial, or a cassette. In some embodiments, the incorporation occurs prior to diagnostic testing.
  • this invention includes a method of increasing the specificity of an assay of a sample, said sample comprising at least one interferent, comprising contacting phosphocellulose to said sample, thereby removing said interferent.
  • said interferent is selected from the group consisting of heme and peroxidase.
  • this invention includes a method of increasing the specificity of an assay of a sample, said sample comprising at least one interferent, comprising contacting a cation exchange material to said sample, thereby removing said interferent.
  • said interferent is elastase.
  • this invention includes a method of extracting at least one interferent from fluids in a lateral flow device, comprising contacting a cation exchange membrane or phosphocellulose to said fluid, thereby removing said interferent.
  • this invention includes a method of enhancing a signal-to-noise ratio in an assay of a biological sample, wherein the sample contains interferents, comprising contacting a cation exchange material or phosphocellulose to said sample, thereby removing said interferents.
  • this invention includes an isolated peptide comprising the amino acid sequence of PFPQANYITY (SEQ ID NO: 1).
  • this invention includes an isolated peptide comprising the amino acid sequence of PMPPLCTSM (SEQ ID NO: 2).
  • the invention described herein also encompasses wound cleansing products, treatment products and wound treatment products.
  • this invention includes a wound cleansing product comprising a gauze or wipe, wherein said gauze or wipe contains a cation exchange material, wherein said cation exchange material removes at least one interferent from said wound.
  • this invention includes a wound cleansing product comprising a gauze or wipe, wherein said gauze or wipe contains phosphocellulose, wherein said phosphocellulose removes at least one interferent from said wound.
  • the invention also encompasses kits, including assay kits.
  • the assay is selected from the group consisting of an enzyme-based assay, an immunoassay, an immunostaining application, an antibody-based assay, a PCR-based assay, a protease-based assay, a diagnostic test, a push through assay and a lateral flow assay.
  • this invention includes a biological assay kit comprising a cation exchange material, wherein said cation exchange material removes at least one interferent from a sample prior to assessment of the sample.
  • this invention includes a biological assay kit comprising phosphocellulose, wherein said phosphocellulose removes at least one interferent from a sample prior to assessment of the sample.
  • this invention includes a biological assay kit comprising a cation exchange material, phosphocellulose and a diagnostic assay device.
  • the diagnostic assay device is selected from the group consisting of a biopsy punch device and a lateral flow device.
  • said interferent is removed prior to an immunostaining or a PCR reaction.
  • said cation exchange material or phosphocellulose is applied to a gauze, cleansing wipe, bead, glass frit, swab, wipe, pad, membrane, a non-woven fiber, a sponge, a filter, or a dressing prior to contact to said sample.
  • FIG. 1 is a standard curve plot for human neutrophil elastase.
  • FIG. 2 is a table of ⁇ g/ml of the concentration of human neutrophil elastase from wound samples.
  • FIG. 3A is a plot graph of the cross-reactivity of specific peptide substrate targets with human elastase.
  • FIG. 3B is a plot graph of the cross-reactivity of specific peptide substrate targets with human elastase.
  • FIG. 3C is a plot graph of the cross-reactivity of specific peptide substrate targets with human elastase.
  • FIG. 4 is a bar graph of the amount of elastase detected in untreated wound fluid and pretreated wound fluid.
  • FIG. 5 is a plot graph of the activity of the peptide HE1 (H 2 N-PFPQANYITYC-OH) (SEQ ID NO: 9) versus specific protease targets.
  • FIG. 6 is a plot graph of the reactivity, as demonstrated by relative fluorescence, of the peptide PAIN1 (NH 2 -E(Edans) PMPPLCTSMK(Dabcyl)-COO11) (SEQ ID NO: 10) with specific protease targets.
  • the top data line indicates ACCUZYME®
  • the next data line indicates PA14
  • the next data line indicates S. pyo BHI.
  • FIG. 7 is a bar graph demonstrating the detection and removal of peroxidases.
  • the left bar (clear) indicates the control and the right bar (filled) indicates P11 treatment.
  • FIG. 8 is a bar graph of the activity of control wound fluid containing bacterial protease versus P11 phosphocellulose-treated wound fluid containing bacterial protease.
  • the left bars (lighter color) indicate control and the right bars (darker color) indicate P11 treatment.
  • FIG. 9 is a bar graph of the activity of control wound fluid containing bacterial protease versus SP sepharose-treated wound fluid containing bacterial protease.
  • the left bars (lighter color) indicate control and the right bars (darker color) indicate P11 treatment.
  • FIG. 10 is a graph of the demonstrating the removal of endogenous peroxidase activity of blood by shifting the pH of the assay conditions to favor the activity of HRP (pH 4.0). The graph demonstrates that at a low pH, HRP strongly reacts with the ABTS substrate whereas the peroxidase activity from blood is minimal.
  • This invention encompasses a method of removing at least one interferent from a sample comprising contacting a cation exchange material or phosphocellulose to said sample, thereby removing said interferent.
  • interferent includes, but is not limited to, peroxidases, elastase, papain, matrix metalloproteases, heme, hemoglobin and viruses.
  • An interferent includes matter in a sample which lowers the signal-to-noise ratio of an assay, likely reducing its specificity.
  • phosphocellulose includes, but is not limited to, p11 resin and p81 paper.
  • cation exchange material includes, but is not limited to, sepharose such as sulfopropyl sepharose and carboxylmethyl sepharose.
  • sample includes, but is not limited to any biological or bodily sample, for example, a sample obtained from or derived from an animal (e.g., a human). “Samples” include, but are not limited to wound, tissue, exudate, urine, saliva, blood, stool, fluid, biopsy and solid samples.
  • an assay includes, but is not limited to, an enzyme-based assay, an immunoassay, an immunostaining application, an antibody-based assay, a polymerase chain reaction (PCR)-based assay, a protease-based assay, a diagnostic test, a push through assay and a lateral flow assay.
  • an enzyme-based assay an immunoassay, an immunostaining application, an antibody-based assay, a polymerase chain reaction (PCR)-based assay, a protease-based assay, a diagnostic test, a push through assay and a lateral flow assay.
  • an enzyme-based assay includes, but is not limited to, an enzyme-based assay, an immunoassay, an immunostaining application, an antibody-based assay, a polymerase chain reaction (PCR)-based assay, a protease-based assay, a diagnostic test, a push through assay and a lateral flow assay.
  • PCR polymerase
  • Human elastase is produced in wound fluid at a variable concentration of approximately 50 ng-60 ⁇ g/1 ml of sample. See Schonfelder, U. et al., “Influence of Selected Wound Dressings on PMN Elastase in Chronic Wound Fluid and Their Antioxidative Potential in Vitro,” Biomaterials, 26(33):6664-6673 (2005).
  • Human elastase is a basic protein with an isoelectric point of 8.77 and a molecular weight of 25,685.71 Da.
  • the enzyme when present in a high concentration in wound fluid (or other tissue samples), can be a problem for antibody, enzyme, or PCR-based diagnostic approaches. In the case of an enzyme-based reporter system that measures a specific protease, elastase can cause false positives, thereby reducing the specificity of such an assay.
  • Human elastase is a well-characterized enzyme with the active center hydrolyzing between the bonds of non-aromatic uncharged amino acids -P 1 -P 1 ′.
  • P 1 is a nonaromatic uncharged amino acid (such as A,V,L,I,S) and P 1 ′ is any amino acid except proline.
  • P 1 is a nonaromatic uncharged amino acid (such as A,V,L,I,S)
  • P 1 ′ is any amino acid except proline.
  • Peroxidases Human fluids contain a number of peroxidases, most notably glutathione peroxidases, myeloperoxidases, and eosinophil peroxidases.
  • Peroxidases have been purified using a variety of methods including ion exchange, concavalin A (Con A), and hydrophobic interaction chromatography. See Anspach, F. B. et al., “High-Performance Liquid Affinity Chromatography with Phenylboronic Acid, Benzamidine, Tri-L-Alanine, and Concanavalin A Immobilized on 3-Isothiocyanatopropyltriethoxysilane-Activated Nonporous Monodisperse Silicas,” Anal.
  • Peroxidases can interfere with assays that use HRP as the reporter enzyme. In addition to these enzymes, we have found that heme in blood can also cause non-specific colorization of the substrate, thereby producing some false positives. Although there is a commercial polyelectrolyte available to remove hemoglobin from diagnostic assays (HEMAGLOBINDTM, Biotech Support Group, NJ, U.S. Pat. No. 5,294,681), our findings indicate that HEMAGLOBINDTM, only removes about 50% of the peroxidase-like interferents in human wound samples.
  • phosphocellulose P11, P81, Whatman plc, Brentford, United Kingdom
  • a cation exchange membrane such as CM, SP
  • cation exchange material such as SP-sepharose beads (GE Life Sciences) will remove elastase and peroxidases with a basic isoelectric point and the P11 or phosphocellulose material will also remove heme from wounds or blood samples that would interfere with the color reactions of an alkaline phosphatase or horseradish peroxidase-based assay.
  • the phosphocellulose binds and removes proteases.
  • peroxidase activity (e.g., of blood) can be removed by shifting the pH of the assay conditions to favor the activity of HRP (for example, to pH of 4.0).
  • HRP strongly reacts with the ABTS substrate whereas the peroxidase activity from blood is minimal.
  • the pH can be shifted to a range of approximately 3-7.5, for example, approximately 3.5-4.5, for example, approximately 4.
  • the invention also encompasses peptides which recognize interferents.
  • suitable peptides include the sequence PFPQANYITY (referred to herein as SEQ ID NO: 1) and the sequence PMPPLCTSM (referred to herein as SEQ ID NO: 2) and/or a modified peptide, for example, one containing one or more conserved amino acid substitutions, and peptides that incorporate or comprise SEQ ID NO: 1 and/or SEQ ID NO: 2 or a modified peptide described herein.
  • peptides include those described herein, as well as those peptides known in the art to undergo modification by interaction with a protein.
  • U.S. patent application Ser. No. 11/036,761, filed Jan. 14, 2005, by Mitchell C. Sanders, entitled A Device for Detecting Bacterial Contamination and Method of Use U.S. patent application Ser. No. 10/502,882, which is the U.S. National Stage of International Application Number PCT/US03/03172 filed on Jan. 31, 2003, and International Application Number PCT/US2004/036469 filed on Nov. 3, 2004, describe such peptides and their teachings are incorporated herein by reference in their entirety.
  • Such peptides described herein can be synthesized from commercial sources, such as New England Peptide (Gardner, Mass.), Sigma-Aldrich, Corp. (St. Louis, Mo.) or Molecular Probes (Eugene, Oreg.), or can be produced (e.g., isolated, purified, or synthesized) using methods known to those of skill in the art.
  • the peptides are specific to interferents, i.e., they recognize interferents significantly more that they recognize other proteins in a sample. In some embodiments, the peptides are highly specific, for example, they recognize an interferent but they do not detectably recognize other enzymes in a sample.
  • a substrate of the invention can include a benzyl ether protecting group bound to one or more of the serine acids on the peptide chain.
  • Protecting groups are chemical groups that are used to protect an amino acid from reacting with a colorimetric component. The use of protecting groups allows for labeling of the same type of amino acid in one peptide with two colorimetric components. For example, one serine group in a peptide can be protected with a benzyl ether group and a second serine, which is not protected, can be reacted with one colorimetric component. The protecting group can be removed and the second serine can be reacted with a different color component, thereby creating a substrate with two different color components.
  • a cysteine (“C”) is added to the carboxyl terminal of the peptide.
  • C cysteine
  • the HE1 peptide when coupled to a reporter, is H 2 N-PFPQANYITYC-OH (SEQ ID NO: 9).
  • amino acids can be appended to the termini of the peptides to introduce the dyes.
  • an “E” is added to the amino terminal and a “K” is added to the carboxyl terminal
  • an “E” is added to the N terminal and a “K” is added to the C terminal, (NH 2 -E(Edans) PMPPLCTSMK(Dabcyl)-COOH) (SEQ ID NO: 10).
  • the peptides of the invention also encompass fragments and sequence variants of the peptides described herein.
  • Variants include a substantially homologous peptide encoded by the same genetic locus in an organism, i.e., an allelic variant, as well as other variants.
  • Variants also encompass peptides derived from other genetic loci in an organism.
  • Variants also include peptides substantially homologous or identical to these peptides but derived from another organism and or D and L isomers (i.e., an ortholog), produced by chemical synthesis, or produced by recombinant methods.
  • the peptide that undergoes modification through interaction with a protein comprises an amino acid sequence, such as one of the sequences listed herein or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity to one of the sequences listed herein, as determined using a sequence comparison program and parameters described herein.
  • the length of the amino acid sequence aligned for comparison purposes is at least 30%, preferably, at least 40%, more preferably, at least 60%, and even more preferably, at least 70%, 80%, 90%, or 100% of the length of the reference sequence.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm.
  • the default parameters of the respective programs can be used.
  • the database searched is a non-redundant database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.
  • the percent identity between two amino acid sequences can be determined by using the GAP program in the GCG software package (available from Acceirys, Inc. of San Diego, Calif., at http://www.accelrys.com, as of Aug. 31, 2001) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
  • the percent identity between two amino acid sequences can be determined using a gap weight of 50 and a length weight of 3. Other preferred sequence comparison methods are described herein.
  • the invention also encompasses peptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a peptide encoded by a nucleic acid molecule of the invention (e.g., the ability to act as a substrate for a protein, e.g., a protein produced by a microorganism). Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a peptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al, “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science, 247(4948):1306-1310 (1990), which is incorporated herein by reference.
  • Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.
  • Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • Amino acids in a peptide of the present invention that are essential for modification of a substrate can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. See Cunningham, B. C. and Well, J. A., “High-Resolution Epitope Mapping of hGH-Receptor Interactions by Alanine-Scanning Mutagenesis,” Science, 244:1081-1085 (1989), which is incorporated herein by reference. The latter procedure introduces a single alanine mutation at each of the residues in the molecule (one mutation per molecule).
  • the invention also includes peptide fragments of the amino acid sequence of the various above-mentioned peptides or functional variants thereof.
  • Useful fragments include those that retain the ability to act as substrates for a protein (e.g., a protein produced by a microorganism.
  • Fragments can be discrete (not fused to other amino acids or peptides) or can be within a larger peptide. Further, several fragments can be comprised within a single larger peptide. In one embodiment, a fragment designed for expression in a host can have heterologous pre- and pro-peptide regions fused to the amino terminus of the peptide fragment and an additional region fused to the carboxyl terminus of the fragment.
  • the peptide can be produced using standard recombinant protein techniques. See Ausubel, F. M. et al., eds., “Current Protocols in Molecular Biology,” (John Wiley & Sons) (1998), the entire teachings of which are incorporated herein by reference). By testing peptide variants with a purified protease, a more specific peptide can be defined, if so desired.
  • Peptides can be synthesized and conjugated with an enzyme (such as horseradish peroxidase (HRP), alkaline phosphatase (AP) or phenyl oxidase (PO)) or simple dye molecule (e.g., blue dye #1) that allows for the determination of both the sensitivity and the specificity of a peptide for a particular microorganism.
  • an enzyme such as horseradish peroxidase (HRP), alkaline phosphatase (AP) or phenyl oxidase (PO)
  • simple dye molecule e.g., blue dye #1
  • a 3-step process can be used: 1) labeling HRP with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) in sodium phosphate buffer, pH 7.5 to produce a maleimide form; 2) conjugation of HRP maleimide to the peptide in phosphate buffer with 5 mM EDTA; and 3) coupling the HRP-peptide to microbeads.
  • SMCC sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • the coupling of the HRP peptide to the micro-beads is performed with the crosslinker, EDC(N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride) in MES buffer.
  • EDC conjugates the carboxyl groups on the bead to the amino terminus of the peptide.
  • blue dye 1 can be synthesized with a maleimide to conjugate directly to the cysteine at the C-terminus of the peptide.
  • the cationic exchange and phosphocellulose materials can be incorporated into a lateral flow device.
  • a lateral flow format provides a simple and rapid point-of-care diagnostic.
  • the device can have four components: a lateral flow strip (1), a conjugate membrane (2), a substrate line (3), and a wicking pad (4).
  • the conjugate pad will likely be a glass microfiber membrane and will be printed with, for example, the HRP-peptide-beads.
  • the glass microfiber slows the flow of the liquid, thereby allowing time for the microbial protease to react with the HRP-peptide-beads.
  • the lateral flow membrane transfers the released HRP to the printed substrate (naphthol) and the wicking membrane at the back of the device acts as a sink to drive the liquid flow through the device.
  • the substrate turns blue and forms a line on the lateral flow membrane.
  • the naphthol substrate can slowly diffuse, preventing the formation of a very distinct line. Dissolving the naphthol in Colloidon (2% nitro-cellulose in amyl acetate) is sufficient to keep the line in place.
  • the materials can be incorporated into the conjugate membrane or incorporated into the swab materials.
  • each chemistry is printed on a separate lateral flow membrane and then three strips can be laminated together in the final device.
  • FIG. 1 It is possible to determine the concentration of elastase in human wound fluids based on a standard curve for the concentration of active elastase in a sample ( FIG. 1 ).
  • the standard curve for human elastase is shown using the Hbt Human Elastase ELISA kit from Hycult Biotechnology (The Netherlands).
  • the human neutrophil elastase used as the standard is from Athens Research & Technology (Athens, Ga.).
  • Human wound fluid was collected on a polyester swab and then extracted with 1.5 ml of phosphate buffered saline (pH 7.5) (designated by swab number) for testing in the Hbt Human Elastase ELISA kit from Hycult following the kit instructions.
  • the elastase standard curve from FIG. 1 was used to convert assay results into ⁇ g/ml of elastase in FIG. 2 .
  • a small aliquot of the fluid was used so that the amount of elastase would be in the range of our standard curve, 0-3 ⁇ g/ml.
  • a few of the very high elastase samples were diluted 1:10 so that they would be in the range as well.
  • the highest amount of elastase obtained from the samples was 60.5 ⁇ g/ml (from sample #106) ( FIG. 2 ).
  • CPI2 peptide sequence EGAMFLEAIPMSIPK (referred to herein as SEQ ID NO: 3)
  • SEQ ID NO: 3 peptide sequence EGAMFLEAIPMSIPK (referred to herein as SEQ ID NO: 3)
  • E8 (peptide sequence EQADALHDQASALK (referred to herein as SEQ ID NO: 4)) and G6 (peptide sequence EAAHQSALQSAK (referred to herein as SEQ ID NO: 5))
  • SAP2 Staphylococcus aureus
  • SAP2 peptide sequence ETKVEENEAIQK (referred to herein as SEQ ID NO: 6)
  • Escherichia coli T2 (peptide sequence EVSRRRRRGGK (referred to herein as SEQ ID NO: 7)
  • T3 (peptide sequence KKASEVSRRRRRGGK (referred to herein as SEQ ID NO: 8)) were also studied.
  • the pathogens were tested with three concentrations of human neutrophil elastase.
  • the peptide substrates were tested in the FRET format with the fluorescent dye Edans attached to one end of the peptide and the quencher Dabcyl attached to the other end of the peptide.
  • amino acids are added to the terminals of the peptides to introduce the FRET assay dye.
  • an “E” is added to the N terminal and a “K” is added to the C terminal.
  • the “E” can be (E-EDANS) and the “K” can be (K-Dabcyl). Cleavage of the peptide substrate by human neutrophil elastase will produce a fluorescent signal.
  • FIG. 3A shows no signal on any of the peptides when tested with 500 ng/ml of elastase.
  • E8 is also known as PAE8.
  • G6 is also known as PAG6.
  • KKAS serves as a linker.
  • FIG. 4 demonstrates the removal of interfering human elastase activity from wound samples by pretreatment with cation exchange resin (such a SP sepharose and phosphocellulose (P11) resins)).
  • cation exchange resin such as SP sepharose and phosphocellulose (P11) resins
  • Wound samples with a range of elastase concentrations were chosen to test removal by SP/P 11 resin treatment of the samples.
  • 5 ⁇ l of the swab samples were treated with 40 ⁇ l of a 50% slurry of SP/P11 (50:50) and 75 ⁇ l of PBS. The samples were incubated for 5 minutes at room temperature and the resin removed by centrifugation.
  • the elastase concentrations before and after SP/P11 treatment were determined using the Hycult ELISA assay and shown as ng of elastase per sample. Untreated and treated samples were diluted such that the results were on the standard curve of the Hycult ELISA assay.
  • FIG. 4 shows efficient removal of human elastase from six wound samples. After pretreating sample 189 with the P11/SP slurry, the amount of detectable elastase was less than 50 ng/sample.
  • a library of peptides was screened to develop peptide sequences that would be specific for bacteria but would not cross react with elastase or another enzyme, papain (that is used in the enzymatic debridement of wounds). Peptide sequences that were specific for both elastase and papain were identified.
  • a peptide specific for elastase was designed from a high throughput peptide library screen.
  • the peptide was tested for activity by conjugating one end to Affigel 10 beads (BioRad) and the other end to HRP reporter enzyme.
  • the sequence of the HE1 peptide used for this assay was H 2 N-PFPQANYITYC-OH) (SEQ ID NO: 9).
  • the activity was measured by detecting the release of HRP from the beads in a spin through assay. Testing was performed with 20 ⁇ l of a 1:10 dilution of HE1-Affigel beads reacted with 5 ⁇ g/ml elastase in 200 ⁇ l total volume for 5 minutes. The supernatant above the beads was tested for HRP release (10 ⁇ l in 490 ⁇ l ABTS substrate) and a clear signal was seen at 405 nm over 5 minutes.
  • PAIN1 a peptide that was found to detect the presence of papain was derived and called PAIN1 ( FIG. 6 ).
  • PAIN1 was designed from a high throughput screen. The peptide was tested in the FRET format with the fluorescent dye Edans attached to one end of the peptide and the quencher Dabcyl attached to the other end of the peptide.
  • the sequence of the PAIN1 peptide used for this assay was NH 2 -E(Edans) PMPPLCTSMK(Dabcyl)-COOH (SEQ ID NO: 10). Cleavage of the peptide substrate by papain will produce a fluorescent signal.
  • the peptide was tested using 5 ⁇ l of a 5 mg/ml solution with 10 ⁇ l of overnight grown bacterial culture or with 10 ⁇ l of 120 mg/ml ACCUZYME®, a papain-containing debriding ointment (DPT Laboratories), in 100 ⁇ l total volume.
  • the bacteria cultures tested were Pseudomonas aeruginosa (PA14), Streptococcus pyogenes (grown in BHI and TSB media), E. coli, Enterococcus faecalis , and Serratia marcesens. The relative fluorescence is shown over one hour. A high response was seen with papain ointment, but there was a lack of specificity since P.
  • aeruginosa and S. pyogenes cultures produce lower but significant responses.
  • the peptide PAIN1 was found to have a strong signal for papain but found to cross-react with microbial proteases.
  • the bacteria that appear to cross-react the most with PAIN1 were Pseudomonas aeruginosa and Streptococcus pyogenes.
  • animal peroxidases, papain, and matrix metalloproteases can also cause problems, particularly when horseradish peroxidase is used as a reporter enzyme.
  • a number of charged (SB6407, ICE, Biodyne C, Biodyne B, P11), uncharged (P4), and hydrophobic (C8 and C18) membranes were screened to determine if they could remove peroxidase activity. Results indicate that the only material successful at removing peroxidases was P11 phosphocellulose. Wound fluids from a pig have considerable peroxidase activity that can be suppressed by pretreatment of the sample with P11 ( FIG.
  • Fluid from a porcine partial thickness wound was collected with a pipet and then the peroxidase activity was measured using the substrate ABTS containing 1 mM hydrogen peroxide. The end point of the blue color was read in a spectrophotometer at 405 nm.
  • the inherent peroxidase activity of one wound sample, sw107 was tested before and after treatment with P11 resin. 40 ⁇ l of a 50% slurry of P11 was added to 75 ⁇ l of PBS and 5 ⁇ l of sw107 and incubated for 5 minutes at room temperature. The resin is removed by centrifugation.
  • the human peroxidase activity is visible as a purple spot that discolors the surface of the membrane. If the human wound samples are pretreated with a slurry of P11/SP resin, then the peroxidase activity is undetectable using the same spot assay that showed the pretreated sample. Specifically, clinical samples 212 and 226 had residual peroxidase activity (left panel) that could be removed after pretreatment with the P11/SP resin (right panel).
  • FIG. 1 demonstrates the detection and removal of human peroxidases from wound fluids.
  • a 50:50 mixture was made of a 40 mg/ml 4 chloro-1-naphthol (Pierce) solution and 2% Collodion in amyl acetate (Electron Microscopy Sciences).
  • a 10 ⁇ l spot of this mixture was placed in the center of a lateral flow membrane (K membrane, Porex) and allowed to dry overnight.
  • K membrane, Porex a lateral flow membrane
  • a 1 ng HRP standard in 10 ⁇ l was placed onto the naphthol spot along with 10 ⁇ l of a 3% hydrogen peroxide solution and a light purple spot develops color.
  • 10 ⁇ l of 1:10 of an artificial wound fluid (AWF) solution was used as a negative control with 10 ⁇ l of a 3% hydrogen peroxide solution.
  • 10 ⁇ l of wound fluid sample extracted from a swab is also placed onto a naphthol spot along with 10 ⁇ l of a 3% hydrogen peroxide solution. These samples are labeled as 212 un and 226 un.
  • the same wound fluid samples that have been treated with SP and P11 for 5 minutes and then filtered, are also tested on naphthol spots in the same manner: 10 ⁇ l of the SP/P11 treated wound fluid sample with 10 ⁇ l of a 3% hydrogen peroxide solution. These samples are labeled as 212 tr and 226 tr.
  • FIGS. 8 and 9 demonstrate that the bacterial proteases added to the wound fluid are not bound by the pretreatment with P11 and SP. It was found that P11/SP removes molecules that reduce the activity of the enzymes, thereby providing more signal after P11/SP treatment. These results indicate that, although P11 and SP can remove very basic proteins such as elastase and peroxidase, in contrast, most neutral or acidic proteins are unaffected by the pretreatment.
  • the graph in FIG. 8 demonstrates that P11 and SP resins did not remove bacterial proteases.
  • a 50% slurry of P11 resin in water was used.
  • a 40 ⁇ l aliquot of P11 slurry was mixed with 10 ⁇ l of overnight grown bacterial supernatant (from Staphylococcus aureus, Pseudomonas aeruginosa , and Enterococcus faecalis ) and 110 ⁇ l PBS.
  • the P11 was given 5 minutes to bind with mixing.
  • the resin was then removed by centrifugation and the bacterial supernatants were tested before and after treatment (with the same dilutions) for activity in the ECI Express Detect system.
  • the released HRP enzyme activity was measured with ABTS substrate.
  • the activity of the bacterial supernatants prior to P11 treatment was normalized to one and the relative activity after P11 treatment is shown in FIG. 8 .
  • the same method was used to demonstrate that SP resins did not remove bacterial proteases.
  • the activity of the bacterial supernatants before and after SP treatment is shown in FIG. 9 in terms of an endpoint OD 405 nm measurement of the ABTS substrate.
  • FIG. 10 demonstrates that the endogenous peroxidase activity of blood can also be removed by shifting the pH of the assay conditions to favor the activity of HRP, an enzyme used in diagnostic assays (for example to pH 4.0). At a low pH, HRP strongly reacts with the ABTS substrate whereas the peroxidase activity from blood is minimal.
  • the invention includes a method of removing peroxidase activity in a sample comprising shifting the pH in said sample, thereby removing said peroxidase activity.

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US9303063B2 (en) 2011-03-18 2016-04-05 Duke University Peptide compounds for suppressing inflammation
JP6158097B2 (ja) 2011-03-18 2017-07-05 デューク・ユニヴァーシティ 炎症を抑制するためのペプチド
US10774363B2 (en) 2015-09-25 2020-09-15 Woundchek Laboratories (Us), Inc. Methods of prediction of wound healing
WO2021252810A1 (fr) 2020-06-10 2021-12-16 Checkable Medical Incorporated Dispositif de diagnostic in vitro

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